The relevance of calcium-binding domains to promoting activities of annexin A2 in calcium oxalate stone formation

Abstract

Annexin A2 (ANXA2) is a Ca2+-binding protein involved in kidney stone disease (KSD) but with unclear mechanism. Herein, five Ca2+-binding domains of ANXA2 were mutated by substituting glutamic acid (E) at positions 53rd (domain I), 96th (domain II) and 247th (domain IV), and aspartic acid (D) at positions 162nd (domain III) and 322nd (domain V) with alanine (A). Recombinant ANXA2 wide type (WT) and mutants (E53A, E96A, D162A, E247A and D322A) were constructed, produced, purified and subjected to multiple crystal and functional assays. Crystal assays revealed that ANXA2 WT increased calcium oxalate monohydrate (COM) crystal size during crystallization and enhanced growth and crystal-cell adhesion phases compared with blank and negative controls. However, crystals exposed to all ANXA2 mutants had comparable or slightly lower parameters compared with controls. Although ANXA2 WT did not affect crystal aggregation, its mutants still showed a lower degree of crystal aggregation. Immunofluorescence staining and Ca2+-binding assay demonstrated that ANXA2 WT had the greatest affinity to COM crystals and free Ca2+ ions, whereas all the mutants showed lower affinity. Taken together, all five Ca2+-binding domains are relevant to the promoting activities of ANXA2 in COM stone formation by interacting with COM crystal surfaces and free Ca2+ ions.